Date of Graduation
5-2016
Document Type
Dissertation (PhD)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Michael C. Lorenz
Committee Member
Jeffrey Actor
Committee Member
Ziyin Li
Committee Member
Kevin Morano
Committee Member
Ambro van Hoof
Abstract
As a commensal colonizer and opportunistic pathogen, Candida albicans is the most clinically important human associated fungus. Systemic infection carries an unacceptably high mortality rate of ~40% in the growing population of immunocompromised individuals. Macrophages are important innate immune cells that limit the niches in the human body in which C. albicans can persist through phagocytic removal. However, following phagocytosis C. albicans readily escapes from the immune cell by differentiating into filamentous hyphae, a process that should be inhibited in the normally acidic phagolysosome. We have shown that C. albicans induces germination by neutralizing the phagolysosome. To better understand this process we compared transcript profiles of cells in conditions that promote alkalinization in vitro to macrophage phagocytosed cells, which revealing an overlapping set of up-regulated genes, including several members of the poorly understood ATO family. This family is greatly expanded in C. albicans relative to other fungi and has been implicated in both ammonia release (Ammonia Transport Outward) and acetate metabolism. I hypothesized that the Ato proteins are important effectors of the pH change in vitro and in macrophages. Deletion of one of the 10 homologs, ATO5, or the over-expression of a dominant negative ATO1G53D allele results in a delay in environmental alkalinization in vitro, a defect in hyphal formation. Further, these strains form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Analysis of an ato5Δ ATO1G53D double mutant strain revealed additive in vitro defects, similar in magnitude to the stp2∆ mutant. Additionally, over-expression of many ATO genes in a wild-type background significantly increases alkalinization and ammonia release, strongly suggesting functional overlap between them. In a complementary approach we examined Ato function in S. cerevisiae Ato proteins as important to weak acid stress tolerance and cytosolic pH homeostasis; revealing that ato mutants are sensitive to weak acid stress and are unable to maintain cytosolic pH homeostasis. This defect was largely dependent upon ScAto1. Taken together, we conclude that Ato proteins are important mediators of the host-pathogen interaction by regulating pH in some host niches.
Keywords
Metabolism, Macrophages, Fungi, Candida albicans, Saccharomyces cerevisiae, acetate, transport